0001] The present invention relates to a method for producing L-methionine by enzyme reaction between a precursor of L-methionine, dimethyl disulfide (DMDS) and hydrogen. It also relates to a two-step process for producing L-methionine by enzyme reaction between a precursor of L-methionine and methyl mercaptan, the latter being obtained by enzymatic DMDS hydrogenolysis with hydrogen.

[0002] Methionine is an essential amino acids the human body and is widely used as an additive for animal feed. It is also used as a raw material for pharmaceuticals. Methionine acts as a precursor of such compounds as choline (lecithin) and creatine. It is also a raw material for synthesis of cysteine ​​and taurine.

[0003] S-adenosyl-L-methionine (SAM) is a derivative of L-methionine and is involved in the synthesis of various neurotransmitters in the brain. L-methionine and / or SAM, inhibit fat accumulation in the body and improves blood circulation in the brain, heart and kidneys. L-methionine can also be used to promote digestion, detoxification and excretion of toxic substances or heavy metals such as lead. It has an anti-inflammatory effect on bone and joint diseases and is also an essential nutrient for the hair, preventing their premature and unwanted fall.

[0004] Methionine is already known to be prepared industrially by chemical pathways from raw materials derived from petrochemicals, as described for example in FR2903690 documents, WO2008006977, US2009318715, US5990349, WO9408957 and JP19660043158. Apart from the fact that these methods of preparation are not part of a process of sustainable development, these chemical methods have the disadvantage of producing a separate equal mixture of two enantiomers L and D.

[0005] totally organic syntheses by bacterial fermentation have been proposed in the literature with the advantage of producing only the L-enantiomer of methionine, as described for example in WO07077041 documents

WO09043372, WO10020290 and WO10020681. Nevertheless, the absence of industrial production on a large scale to date, suggests that performance and / or production costs of these processes are still inadequate.

[0006] chemical / biological processes mixed just been successfully developed jointly by the company CJ Cheil-Jedang and the Applicant, in which a precursor of L-methionine is produced by bacterial fermentation and then reacts enzymatically with méthylmercatan for producing exclusively L-methionine (see WO2008013432 and / or WO2013029690). These methods although high performance require the synthesis of methyl mercaptan site, which itself requires the synthesis of hydrogen by steam reforming of methane, the synthesis of hydrogen sulfide by hydrogenation of the sulfur and its synthesis from methanol and hydrogen sulphide, c '

[0007] There therefore remains a need for producing L-methionine by a mixing process wherein the equipment required for the synthesis of methyl mercaptan will be less than for a synthesis from hydrogen, hydrogen sulfide and methanol. It is in this context that the present invention takes place.

[0008] The present invention provides in effect replacing the methyl mercaptan in the process summarized below (WO2008013432 and / or WO2013029690) with dimethyl disulfide (DMDS):

MeSH

O-Acélyihomosénne fermentation Catalysis enzymattque

L-Methionine Step 1 (OAHS) £ ape 2

[0009] The methyl mercaptan (MeSH) is here used directly in the second step. The present invention proposes to substitute the methyl mercaptan by the enzymatic hydrogenolysis product of dimethyl disulfide in a preliminary step or combine together in a "one pot" reaction, in which glucose and DMDS produce L-methionine.

[0010] Regarding the synthesis of methyl mercaptan from dimethyl disulfide, can be found in the prior art the following items.

[0011] The EP0649837 patent application proposes a methyl mercaptan synthesis process by catalytic hydrogenolysis, with transition metal sulfides, the dimethyl disulfide with hydrogen. This method, although effective, requires relatively high temperatures of the order of 200 ° C to obtain industrially interesting productivities.

[0012] The skilled person also knows that it is possible to prepare methyl mercaptan by acidification of an aqueous solution of sodium methyl mercaptide (ChbSNa). This method has the major drawback of generating large amounts of salts such as sodium chloride or sodium sulphate, depending on whether hydrochloric acid is used or sulfuric acid. The aqueous salt solutions are often very difficult to treat and traces of malodorous products remaining are that this method is hardly feasible industrially.

[0013] It has now been found that could prepare the methyl mercaptan by enzymatic reduction of dimethyl disulfide (DMDS) during a preliminary step in the synthesis of L-methionine and it has also been found, surprisingly that we could achieve this enzymatic reduction of DMDS during the synthesis of L-methionine.

[0014] Thus, the present invention does relates to a process for preparing L-methionine similar to that proposed in international applications WO2008013432 and / or WO2013029690 and allows to overcome or at least to decrease, handling methylmercaptan, generating said methyl mercaptan in an enzymatically catalyzed reaction of DMDS, just before the use of said methyl mercaptan in the synthesis of methionine or generating said methyl mercaptan in an enzymatically catalyzed reaction of DMDS in situ in the reactor synthesis of L-methionine.

[0015] More particularly, the present invention has as its first object the method for preparing L-methionine, comprising at least the steps of:

a) preparing a mixture comprising:

1) dimethyl disulfide (DMDS)

2) a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

3) a catalytic amount of enzyme catalyzing the reaction of disulfide bond reducing said amino acid carrying a thiol group or thiol group of said peptide,

4) hydrogen,

5) a catalytic amount of enzyme catalyzing the reaction of hydrogen reduction,

6) a catalytic amount of a cofactor common to the two enzymes of the catalyst system (dehydrogenase and reductase),

b) conduct the enzyme reaction to form methyl mercaptan (CH3-S), c) adding a precursor of L-methionine and converting said precursor with methyl mercaptan formed in step b) and

d) recovering and optionally purifying the formed L-methionine.

[0016] The components of step a) above can be added in different orders (the order of addition in step a) is not restrictive). In one embodiment of the invention, the amino acid carrying a thiol group and / or peptide bearing a thiol group may be in the form of disulfide said amino acid and / or said peptide, respectively, e.g. glutathione as glutathione disulfide.

[0017] In general, the enzyme catalyzing the reduction of the disulfide bridge formed between two equivalents of said amino acid carrying a thiol group or thiol group of said peptide is a reductase enzyme. The term "reductase" is used in the following description for the explanation of the present invention. Similarly, the enzyme catalyzing the hydrogen reduction reaction is generally referred hydrogen dehydrogenase, the term "dehydrogenase" is selected from the following description for the explanation of the present invention.

[0018] Of the common cofactors for enzymes catalyzing both reduction and dehydrogenation (reductase and dehydrogenase) include as non-limiting examples cofactors flavin, nicotine and cofactors. We prefer to use the cofactor nicotinic especially the nicotinamide adenine dinucleotide (NAD), or better yet the nicotinamide adenine dinucleotide phosphate (NADPH). Cofactors listed above are advantageously used in their reduced forms (e.g., NADPH, H +) and / or their oxidized forms (by

example NADP +), that is to say, they may be added in these reduced forms and / or oxidized, in the reaction medium.

[0019] The organization and order of additions of components 1) to 6) in step a) can be achieved in different ways. The enzymatic reaction in step b) is initiated by the addition of one of the components of the catalytic system of the mixture of step a): either an enzyme or one of the compounds added in stoichiometric quantity (or disulfide reducing organic compound ) is one of the compounds added in a catalytic amount (amino acid carrying a thiol group or thiol group to the peptide or disulfide corresponding to said thiol or said peptide or alternatively cofactor).

[0020] Thus, and according to one embodiment of the present invention, the method for preparing L-methionine comprises at least the steps of:

a ') preparing a mixture comprising:

• dimethyl disulfide (DMDS)

• a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

• a catalytic amount of reductase enzyme corresponding to said amino acid carrying a thiol group or thiol group in said peptide,

• a catalytic amount of NADPH

b ') adding hydrogen with a catalytic amount of hydrogen-dehydrogenase enzyme,

c ') carrying out the enzyme reaction to form methyl mercaptan (CH 3 SH), d') converting a precursor of L-methionine with the methyl mercaptan formed in step c '), and

e ') recovering and optionally purifying the formed L-methionine.

[0021] According to the method of the invention, methyl mercaptan, generally formed in a gaseous state, is then directly contacted with a methionine precursor as described hereinafter.

[0022] The method of synthesis of L-methionine according to the invention is first based on the enzymatic reduction of dimethyl disulphide with hydrogen, according to the following reaction:

[0023] It has now been discovered that this reaction is readily catalyzed by the enzyme system implementing a thiol group to amino acid or a thiol group in the peptide, e.g., glutathione, as a complex (amino acid or peptide) / corresponding reductase enzyme, regenerated with hydrogen, as described in Figure 1 herein.

[0024] Thus according to the Figure 1 illustration, the peptide (example shown glutathione) reduced dimethyl disulfide to methyl mercaptan by transforming into disulfide-bonded peptide (shown glutathione disulfide). The reductase enzyme ( "glutathione reductase" shown, EC 1 .8.1 .7 or EC 1 .6.4.2) regenerates the peptide (glutathione) and the same enzyme is regenerated by a redox enzyme complex known to those skilled the art, such as NADPH / NADP + complex (nicotine adenine dinucleotide phosphate (reduced form and oxidized form)). In its turn NADP + to NADPH is regenerated by means of the enzyme "Hydrogen dehydrogenase" (EC 1 .5 .12.1) with hydrogen. The proton released by the hydrogen s' after reaction with NADPH and function mercaptide formed becomes a mercaptan function.

[0025] According to one embodiment particularly adapted, the system glutathione / glutathione disulfide associated with the glutathione reductase enzyme of the present invention allows to reduce the DMDS methyl mercaptan.

[0026] Glutathione is a tripeptide widely used in biology. This species in reduced form (GSH) and oxidized (glutathione disulfide) forms an important redox couple in the cells. So glutathione is vital to remove heavy metals organizations. For example, the application WO05107723 describes a formulation in which glutathione is used to form a chelate preparation, patent US4657856 teaches that glutathione also used to destroy peroxides such ΙΉ2Ο2 H2O via glutathione peroxidase. Finally glutathione also reduces disulfide bonds in proteins (Rona Chandrawati, "Triggered Cargo Release Encapsulated by Enzymatic Catalysis in Capsosomes" Nano Lett (1 201), Vol. 1 1, 4958-4963).

[0027] According to the method of the invention, a catalytic amount of amino acid carrying a thiol group or thiol group to peptide, is implemented for the production of methyl mercaptan from dimethyl disulphide.

[0028] Among the amino acids thiol group of carriers used in the process of the present invention include, by way of non-limiting examples the cysteine ​​and homocysteine. The redox enzyme systems used can regenerate the catalytic cycle in the same way, these cases are in the system cysteine ​​/ cystine reductase EC 1 .8.1 .6, homocysteine ​​and / homocysteine ​​reductase.

[0029] It may be advantageous to use homocysteine ​​as this amino acid may be prepared from OAHS (precursor of L-methionine), hydrogen sulfide (HS) and the enzyme catalyzing the reaction leading to methionine. Thus, a very small amount of S h in the reaction medium, creates in situ the cycle equivalent to that of glutathione.

[0030] Among the thiol group of bearing peptides used in the process of the present invention include, by way of non-limiting examples glutathione and thioredoxin. Glutathione system / glutathione reductase, described above, can thus be replaced by the thioredoxin system (CAS no. 52500-60-4) / thioredoxin reductase (EC 1 .8.1 .9 or EC 1 .6.4.5).

[0031] The glutathione and glutathione system / glutathione reductase are especially preferred for the present invention, due to the ease of supply and the costs thereof.

[0032] In the method according to the invention, hydrogen can be added to the reaction medium by any means known to those skilled in the art, for example by bubbling in the reaction medium which is preferably an aqueous-organic reaction medium. The hydrogen pressure in the reactor corresponds to the pressure of the reaction medium itself, which is defined below.

[0033] The enzyme used is hydrogen dehydrogenase enzyme, also well known to those skilled in the art.

[0034] In the method according to the invention, in the case where the enzymatic reduction of DMDS is carried out in a separate reactor for the synthesis of L-methionine, only the DMDS and hydrogen are used in stoichiometric amount, all other components (glutathione, cofactor (e.g., NADPH) and the two enzymes) are used in catalytic amount. In the case where the enzymatic reduction reaction of DMDS

is done with the synthesis of L-methionine in a single reactor, said "one pot", the precursor of L-methionine is added in stoichiometric amount, while the additional reagents for this synthesis such as pyridoxal phosphate ( PLP) and the specific enzyme for this reaction are added in catalytic amounts.

[0035] The concentrations of pyridoxal phosphate and specific enzyme preferred precursor are those we can find in international applications WO2008013432 and / or WO2013029690.

[0036] The benefits synthesizing by enzymatic catalysis of methyl mercaptan from dimethyl disulphide are numerous, both in the case of two successive stages or "one pot" method. These benefits include the ability to work in aqueous or aqueous-organic solution, under very mild conditions of temperature and pressure and pH conditions close to neutrality. All these conditions are typical of a process called "green" or "sustainable" and are completely compatible with the preparation of L-methionine, as described in international applications WO2008013432 and / or WO2013029690.

[0037] Another advantage when the process uses dimethyl disulfide is as methyl product, which is in the gaseous state under the reaction conditions, leaves the reaction medium as and when it is formed, possibly accompanied hydrogen that has not reacted. It can therefore be used directly to the reactor outlet in a downstream application if unreacted hydrogen does not interfere therein.

[0038] The methyl mercaptan may be used directly at the outlet of the reactor in the synthesis of L-methionine, as described for example in WO2008013432 and / or WO2013029690, that is to say from e.g. O- acetyl-L-homoserine or O-succinyl-L-homoserine and enzymes such as O-acetyl-L-homoserine sulfhydrylase or Ο-succinyl-L-homoserine sulfhydrylase respectively.

[0039] If the art will easily separate hydrogen unconverted methyl mercaptan. Methyl mercaptan may also be easily liquefied cryogenic e.g. if it is desired to isolate or separate.

[0040] The exit gas containing hydrogen and methyl mercaptan may, if desired and if necessary, be recycled into the first reactor (enzymatic reduction of DMDS) after passing through the second reactor (synthesis of L-methionine) if méthylnnercaptan was not completely converted to L-methionine. The method of the invention thus discloses a method for synthesizing L-methionine in 2 successive enzymatic steps from a precursor of L-methionine and DMDS.

[0041] It is also possible to carry out the synthesis of L-methionine in a single reactor. In this case is added to the enzyme reduction system of DMDS (step a) above) all reagents required for the synthesis of L-methionine and the reactor is closed to prevent the departure of the methyl mercaptan formed by enzymatic reduction in situ DMDS. Methyl mercaptan reacts with the precursor L-methionine to give L-methionine. The method according to the present invention thus discloses a direct synthesis process of L-methionine from a precursor of L-methionine and DMDS, as shown in Figure 2 attached, ie synthesis from OAHS, DMDS and hydrogen.

[0042] Dimethyl disulphide (DMDS) can be produced on a different site from methyl mercaptan and an oxidant such as oxygen, sulfur or oxygenated water for example, or from dimethyl sulfate and sodium disulfide. DMDS can also come from a source of "disulfide Oils" (DSO) purified for example by reactive distillation as described in WO2014033399 application.

[0043] The reduction in enzymatic catalysis of DMDS can be considered as a method to prevent the transportation of methyl mercaptan production site by industrial existing channels, to its site of use, if they are different. In fact, methyl mercaptan is a gas at room temperature, highly toxic and smelly which greatly complicates its transportation already highly regulated unlike DMDS. Thus DMDS can be used to produce methyl mercaptan directly on the site of use thereof in the synthesis of L-methionine, thereby further reducing the disadvantages of toxicity and odor of the product, and industrial risks attached thereto.

[0044] In the case of the synthesis process in two successive steps, DMDS being consumed in the reaction and the outgoing methyl mercaptan from the reaction medium as and when it is formed, with or without unconverted hydrogen, no product s' accumulates in the assumption of a continuous hydrogen supply and DMDS.

It is not necessary to recycle the catalyst system saw the products in and out of the reactor.

[0045] According to one embodiment, the method according to the invention comprises the preparation of methyl mercaptan by enzymatic reduction of DMDS, and then reacting said methyl mercaptan formed with a precursor of L-methionine to give L-methionine. In this case, the method according to the invention comprises at least the following steps:

Step 1: preparing a precursor of L-methionine, for example, by bacterial fermentation of glucose (see WO2008013432 and / or WO2013029690) Step 2: Enzymatic reduction of DMDS in a reactor R1 with methyl mercaptan formation and possibly the unconverted hydrogen, exiting said reactor R1 (steps a) to c) above),

Step 3: Enzymatic synthesis of L-methionine in a reactor R2 with the precursor of step 1 and methyl mercaptan in Step 2 (step d) above),

Step 4 (optional): recycling of unconverted hydrogen to Step 2 and recycling of methyl mercaptan to Step 2 or Step 3, and

Step 5: Recovery and optionally purification of the formed L-methionine (step e) above).

[0046] In Step 1, we find the field conditions used in international applications WO2008013432 and / or WO2013029690.

[0047] For Step 2, the reaction temperature is within a range from 10 ° C to 50 ° C, preferably between 15 ° C and 45 ° C, more preferably between 20 ° C and 40 ° C.

[0048] The pH of the reaction may be between 5 and 9, preferably between 6 and 8.5, preferably between 6 and 8, and most preferably between 7.0 and 8.0. So Most preferably, one will choose the pH of a medium buffered to a pH value between 7.5 and 8.0. According to another preferred embodiment, the pH is chosen to a buffered medium at a pH value between 6.5 and 7.5.

[0049] The pressure used for the reaction can range from a reduced pressure relative to the atmospheric pressure to several bars (several hundred kPa), depending on the reagents used and the equipment used. Preferably, a pressure from atmospheric pressure will be used at 20 bar (2 MPa) and more preferably it will work under a pressure ranging from atmospheric pressure to 3 bars (300 kPa).

[0050] In Step 3, reference to international application WO2013029690 for ideal conditions

[0051] According to another embodiment (another variant), the method according to the present invention is carried out in a single reactor ( "one pot"), and in this case comprises at least the following steps:

Step 1 ': preparing a precursor of L-methionine, for example, by bacterial fermentation, including but not limited to glucose fermentation (similar to Step 1 above),

Step 2 ': enzymatic reduction of DMDS in a reactor R1 with in situ formation of methyl mercaptan and joint enzymatic synthesis of L-methionine in the same reactor with the precursor obtained in step 1',

Step 3 (optional): recycle loop hydrogen and méthlymercaptan in the reactor R1, at the step 2 ', and

Step 4 ': recovering and optionally purifying the formed L-methionine (step e) above).

[0052] In Step 1 ', we find the field conditions used in international applications WO2008013432 and / or WO2013029690.

[0053] In Step 2 ', the operating conditions are as follows.

[0054] The reaction temperature is within a range from 10 ° C to

50 ° C, preferably 15 ° C to 45 ° C, preferably from 20 ° C to 40 ° C.

[0055] The pH of the reaction is advantageously between 6 and 8, preferably between 6.2 and 7.5.

[0056] Preferably, the pressure used for the "one pot" reaction can range from a reduced pressure relative to the atmospheric pressure to several bars (several hundred kPa), depending on the reagents used and the equipment used. Preferably, a pressure from atmospheric pressure will be used at 20 bar (2 MPa) and more preferably it will work under a pressure ranging from atmospheric pressure to 3 bars (300 kPa).

[0057] The molar ratio DMDS / L-methionine precursor is between 0.1 and 10, usually between 0.5 and 5, and preferably the molar ratio is the stoichiometry (molar ratio = 0.5). but may be higher if this is beneficial for the kinetics of the reaction.

[0058] In one or other of the process according to the invention variants, it may be carried out batchwise or continuously in a reactor made of glass or metal depending on the operating conditions and reagents used. According to one embodiment, the method of the invention is a semi-continuous process in which hydrogen is added as and when it is consumed in the reaction.

[0059] In one or the other variants of the method according to the invention, the molar ratio hydrogen / DMDS is ideal stoichiometry (molar ratio = 1) but may range from 0.01 to 100 if those skilled in the art there is any interest such as continuous addition of hydrogen, so as DMDS is introduced from the beginning into the reactor. Preferably this molar ratio is chosen between 1 and 20 globally on the entire reaction.

[0060] The hydrogen that is not converted may be recycled to the reactor outlet to the inlet to total exhaustion. One can also consider a loop with hydrogen and methyl, until hydrogen has completely converted to DMDS. In this configuration, sorite in the gas from the reactor R2 (or of the reactor, where the reaction is conducted in "one pot") almost exclusively contain methyl mercaptan.

[0061] The elements present in a catalytic amount in the mixture prepared in step a) above (amino acid carrying a thiol group or thiol group to peptide, or disulfide corresponding to said amino acid or said peptide , reductase enzyme, dehydrogenase enzyme, cofactor (e.g., NADPH)) are readily available commercially or may be prepared according to techniques well known to those skilled in the art. These different elements may be in solid or liquid form and may most preferably be dissolved in water to be used in the method of the invention. The enzymes used can also be grafted on a support (if the supported enzymes).

[0062] The aqueous solution of enzyme complex comprising the amino acid or peptide can also be reconstituted by methods known to those skilled in the art, for example by permeabilization of cells which contain these elements. This aqueous solution having a composition given in Example 1

the following may be used in weight contents of between 0.01% and 20% based on the total weight of the reaction medium. Preferably we use a content of between 0.5% and 10%.

[0063] The concentrations of pyridoxal phosphate and specific enzyme preferred precursor L-methionine are those that we can find in international applications WO2008013432 and / or WO2013029690.

[0064] the invention will be better understood with the following non-limiting examples in relation to the scope of the invention. All tests listed below were performed under anaerobic conditions.

EXAMPLE 1: A process in two successive steps

[0065] In a reactor R1 containing 150 mL of buffered aqueous solution at pH 7.8, are introduced 10 mL of glutathione enzyme complex (Aldrich). The enzyme complex solution contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH and 200 U hydrogen dehydrogenase enzyme. The reaction medium is brought to 35 ° C with mechanical stirring. A first transaction is made t = 0. Thereafter, the dimethyl disulfide (9.4 g, 0.1 mol) was placed in a buret and added dropwise into the reactor.

[0066] At the same time, a hydrogen stream 4 Lh ~ 1 (measured under normal conditions of temperature and pressure) is introduced by bubbling into the reactor. The reaction is carried out at atmospheric pressure.

[0067] An analysis by gas chromatography of gas leaving the reactor shows almost essentially the presence of hydrogen and methyl mercaptan (traces of water). DMDS and hydrogen (molar ratio hydrogen / DMDS on the entire reaction = 10.7) is added in 6 hours and a final analysis by gas chromatography of the reaction mixture confirms the absence of methyl mercaptan that was expelled from the reactor by the excess hydrogen. These gas outlet of the reactor R1 are sent directly to the reactor R2.

[0068] In parallel, in the second reactor R2 containing 75 mL of phosphate buffer 0.1 mol L "1 at pH 6.60, were introduced 5 g of O-acetyl-L-homoserine (OAHS) (Ο- acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride according Sadamu Nagai, "Synthesis of O-acetyl-L-homoserine," Academy

Press, (1971), vol.17, p. 423-424. The solution was heated at 35 ° C with mechanical stirring.

[0069] Before commencement of the reaction, a sample (t = 0) 1 mL of the reaction medium is performed. A pyridoxal phosphate solution (1; 6 mmol, 0.4 g) and the enzyme O-acetyl-L-homoserine sulfhydrylase (0.6 g) are dissolved in 10 mL of water and then added to the reactor.

[0070] Methyl mercaptan is introduced via the reaction of the reactor R1 and advantageously driven by the excess hydrogen, or when hydrogen is in stoichiometric or sub-stoichiometry relative to the DMDS Methyl mercaptan is advantageously driven by a current inert gas, for example a stream of nitrogen. The reaction begins. The formation of L-methionine and the disappearance of the OAHS followed by HPLC. The gas outlet of the reactor R2 are trapped in an aqueous sodium hydroxide solution (sodium hydroxide) at 20%. Analyzes show that the OAHS was converted to 42% in L-methionine and that excess DMDS was converted to methyl mercaptan found in the trap soda.

EXAMPLE 2 Process "one pot"

[0071] In a reactor containing 150 ml of phosphate buffer 0.2 mol L "1 at pH 7, are introduced 10 mL of the enzyme complex, 5 g (31 mmol) of O-acetyl-L-homoserine (OAHS, ΓΟ-acetyl-L-homoséhne was synthesized from L-homoserine and acetic anhydride according Sadamu Nagai, "Synthesis of O-acetyl-L-homoserine," Academic Press, (1971), vol.17, p. . 423-424) the enzyme of the complex solution contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH, 200 U hydrogen dehydrogenase enzyme, 0.4 g (1, 6 mmol) of pyridoxal phosphate and 0.6 g of O-acetyl-L-homoserine sulfhydrylase.

[0072] The reaction medium is brought to 35 ° C with mechanical stirring. A first sample t = 0 is made. Thereafter dimethyl disulfide (3 g, 32 mmol) was placed in a buret and added dropwise with a rate of 4 liter / h of hydrogen was introduced; the reaction begins. The reaction was followed by HPLC to see the disappearance of the OAHS and formation of L-methionine. After 6 hours, 12% of OAHS have been converted into L-methionine showing the ability to produce L-methionine by a "one-pot" process from a precursor of L-methionine, DMDS and hydrogen.

EXAMPLE 3 Method "one pot"

[0073] A reactor containing 70 ml of phosphate buffer 0.1 mol L "1 at pH 6.8, are introduced 10 mL of the enzyme complex, 5 g (31 mmol) of O-acetyl-L-homoserine ( OAHS O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydridre according Sadamu Nagai, "Synthesis of O-actetyl-L-homoserine", Academic Press, (1971), vol. 17, p. 423-424).

[0074] The complex of enzyme solution contains: 200 mg of glutathione (0.65 mol), 500 U of glutathione reductase, 100 mg of NADPH (0.13 mol), 50 U dehydrogenase hydrogen, 400 mg (1, 6 mmol) of pyridoxal phosphate, 2 g of O-acetyl-L-homoserine and 0.6 g of O-acetyl-L-homoserine sulfhydrylase.

[0075] The hydrogen dehydrogenase is obtained from microorganism culture (according to Biller et al., "Fermentation Hyperthermophiler Mikro-organismen am Beispiel von Pyrococcus furiosus" Shaker Verlag, Maastricht / Herzogenrath, 2002) using techniques well known to the skilled person.

[0076] The reaction medium is brought to 35 ° C under mechanical stirring and nitrogen flushing. A first sample is taken at t = 0. Thereafter, 20 g (0.22 mol) of dimethyl disulfide are added using a syringe. At the same time, 4 Lh ~ 1 hydrogen (measured under normal conditions of temperature and pressure) are introduced by bubbling into the reaction medium. The then initiated reaction is performed at atmospheric pressure for 18 hours. The reaction was followed by HPLC to see the disappearance of OAHS and formation of L-methionine. At the end of the reaction 27% of OAHS have been converted into L-methionine showing the ability to produce L-methionine by a method "one pot" from a precursor of L-methionine, DMDS and '

CLAIMS

A process for preparing L-methionine, comprising at least the steps of: a ') preparing a mixture comprising:

1) dimethyl disulfide (DMDS)

2) a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

3) a catalytic amount of enzyme catalyzing the reaction of disulfide bond reducing said amino acid carrying a thiol group or thiol group of said peptide,

4) hydrogen,

5) a catalytic amount of enzyme catalyzing the reaction of hydrogen reduction,

6) a catalytic amount of a cofactor common to the two enzymes of the catalyst system (dehydrogenase and reductase),

b) conduct the enzyme reaction to form methyl mercaptan (CH3-S), c) adding a precursor of L-methionine and converting said precursor with methyl mercaptan formed in step b) and

d) recovering and optionally purifying the formed L-methionine.

The method of claim 1 comprising at least the steps of:

a ') preparing a mixture comprising:

• dimethyl disulfide (DMDS)

• a catalytic amount of amino acid carrying a thiol group or thiol group to peptide,

• a catalytic amount of reductase enzyme corresponding to said amino acid carrying a thiol group or thiol group in said peptide,

• a catalytic amount of NADPH

b ') adding hydrogen with a catalytic amount of hydrogen-dehydrogenase enzyme,

c ') carrying out the enzyme reaction to form methyl mercaptan (CH 3 SH), α ") converting a precursor of L-methionine with the methyl mercaptan formed in step c'), and

e ') recovering and optionally purifying the formed L-methionine.

3. The method of claim 1 or claim 2, wherein methyl mercaptan is directly contacted with a methionine precursor.

4. A method according to any preceding claim, wherein the thiol-bearing amino acid or thiol group of carrier peptide is selected from cysteine, homocysteine, glutathione and thioredoxin.

5. A method according to any preceding claim, wherein the precursor of L-methionine is selected from O-acetyl-L-homoserine and O-succinyl-L-homoserine.

6. A method according to any preceding claim, wherein hydrogen is added by bubbling into the reaction medium.

7. A method according to any preceding claim comprising the preparation of methyl mercaptan by enzymatic reduction of DMDS, and then reacting said methyl mercaptan formed with a precursor of L-methionine to give L-methionine.

8. The method of claim 7 comprising at least the following steps: Step 1: preparing a precursor of L-methionine,

Step 2: Enzymatic reduction of DMDS in a reactor R1 with methyl mercaptan formation and possibly the unconverted hydrogen, exiting said reactor R1,

Step 3: Enzymatic synthesis of L-methionine in a reactor R2 with the precursor of step 1 and methyl mercaptan in Step 2,

Step 4 (optional): recycling of unconverted hydrogen to Step 2 and recycling of methyl mercaptan to Step 2 or Step 3, and

Step 5: Recovery and optionally purification of the formed L-methionine.

9. A method according to any one of claims 1 to 6, wherein the synthesis of méthylnnercaptan from DMDS and synthesis of L-methionine from said méthylnnercaptan is carried out in a single reactor.

10. The method of claim 9, comprising at least the following steps: Step 1 ': preparing a precursor of L-methionine,

Step 2 ': enzymatic reduction of DMDS in a reactor R1 with in situ formation of methyl mercaptan and joint enzymatic synthesis of L-methionine in the same reactor with the precursor obtained in step V,

Step 3 (optional): recycle loop hydrogen and méthlymercaptan in the reactor R1, at the step 2 ', and

Step 4 ': recovery and optionally purification of the formed L-methionine.

11. A method according to any preceding claim, carried out batchwise or continuously.

12. A method according to any preceding claim, wherein the molar ratio hydrogen / DMDS ideal varies from 0.01 to 100, preferably between 1 and 20 on the entire reaction.

13. A method according to any preceding claim, wherein the molar ratio DMDS / L-methionine precursor is between 0.1 and 10, usually between 0.5 and 5, and preferably the molar ratio is the stoichiometry (molar ratio = 0.5).

14. A method according to any preceding claim, wherein the reaction temperature is within a range from 10 ° C to 50 ° C, preferably 15 ° C to 45 ° C, preferably from 20 ° C to 40 ° C.